Infertility is a significant medical problem affecting a large proportion of the population. About half of the causes of infertility arise in the male (generally the sperm or seminal fluid) and usually no specific cause can be identified, even after thorough evaluation. Several diagnostic tests for such evaluations have been developed and provide some useful information. Generally, studies of patients having unexplained infertility using these tests have reported defects in capacitation and sperm motion characteristics, binding of spermatozoa to the zona pellucida, acrosome reaction, acrosin activity of spermatozoa, and the ability of the spermatozoa to penetrate zona-free oocytes (Mackenna et al, 1995). However, only when such tests identify one or more abnormalities can more specific and cost-effective treatment regimens be instituted (Chuang et al, 1998).
An important clinical test for evaluation of male fertility uses the acrosome reaction whereby acrosin and other acrosome enzymes in sperm are released by simulation of contact with an egg or by actual contact with an egg. Almost all methods for detecting the acrosome reaction monitor a morphological change during the acrosome reaction. The most widely used methods utilize optical microscopy, wherein spermatozoa are visualized after staining. Different chemical agents such as calorimetric dyes and fluorophore labeled lectins and antibodies may be used. However the results of these tests often differ depending on the characteristics of the different agents used.
The problems of acrosome reaction tests arise from several factors, including the timing of capacitation, the reaction medium, the inducing agents, and the timing of reaction. Furthermore, the detection methods may significantly impact the final result. For example, a stain in the outer acrosome membrane typically gives a different result than a stain in the inner acrosome membrane. Perhaps the most reliable method for detection of the AR is electron microscopy. Unfortunately this procedure cannot be used routinely because it is expensive and labor-intensive. Most present methods utilize an optical microscope, wherein spermatozoa are stained for the visualization of their acrosomal status. Membrane changes in the spermatozoa lead to loss of the acrosomal cap, which may be determined morphologically. However, these traditional morphological methods still are labor-intensive and generate unreliable results. More accurate and convenient methods are greatly needed for detecting AR.
Of course, more specific chemical details of the AR beyond the morphological changes are known and might be relied on for diagnosing causes of male infertility. An early development in this field has been the use of calcium ionophores to stimulate morphological changes that can be observed. A recent study of zona-induced AR (ZIAR) had found that such induction had a high predictive value for IVF results (Esterhuizen A D, 2001). Physiologically, AR is induced by zona pellucida protein and followed by the liberation of several acrosomal enzymes and other constituents that facilitate penetration of the zona. Although the AR is an important step during fertilization, this sperm function generally is not diagnosed correctly and easily by present methods. Generally, most AR studies to date have focused on the morphological change by using chemical (Ca2+ ionophore A23187). Two major problems to date prevent widespread adoption of these techniques: 1) (Ca2+) induced AR responses do not have the same clinical values as ZIAR (Franken D R et al, 1997); and 2) AR detection by morphological changes is subjective and labor-intensive.
Biochemical tests have been designed around assay of AR releasing acrosin and other acrosomal contents. The acrosin protein has been chosen as a marker to detect AR because it accounts for almost 20% of the total acrosomal protein. Unfortunately, despite many efforts, a reliable assay method using acrosin activity to indicate AR has not been suitably commercialized. One reason for this lack of good success is that acrosin is inhibited by two different trypsin inhibitors that exits in human seminal plasma (inhibitors (HUSI) I and II as described by E. Fink, et. Al, 1971 and H. Schiessler, et al, 1974). Both inhibitors greatly interfere with acrosin activity. A second assay for acrosin activity is the photometric enzyme method, wherein a sperm is treated with triton X-100 for lysis and to convert proacrosin to acrosin. Although this method could measure total amount of acrosin activity, it generally does not determine the amount of true acrosin activity from acrosome reacted human sperm.
Other related tests used in the modern assisted reproductive technologies field include basic semen analysis, computer-assisted evaluation of sperm motion characteristics, inducibility of the acrosome reaction, and bioassays that assess gamete interaction, which includes the hemizona assay and sperm-hamster egg penetration assay (Oehninger et al., 1997). However, the hemizona assay is too expensive, since this assay requires human egg, to become routine bioassay. And the sperm-hamster egg penetration assay needs to be implemented before its introduction as a routine clinical tool (Oehninger et al., 1997). Thus, a convenient and accurate assay that can assess the spermatozoa-oocyte interaction is necessary for today's infertility clinical therapies.
More speculation in this area considers possible use of the zona pellucida glycoproteins, which play an exclusive role in mediating the binding between spermatozoa and oocyte. ZP3 mediates an initial binding (Leyton and Saling, 1989; Aarons, et al., 1991; Macek et al., 1991) and ZP2 possibly mediates a secondary binding of spermatozoa to zona pellucida (Bleil and Wassarman, 1980; Bleil et al., 1988). The primary binding is mediated by ZP3 and its specific receptors located on the sperm plasma membrane. The zona pellucida glycoproteins are highly glycosylated and possess both Asn- (N-) linked and Ser/Thr- (O-) linked oligosaccharides. Different carbohydrates on ZP3, such as galactose in an alpha-linkage, N-acetylglucosamine in a beta-linkage, were suggested as complementary sperm receptors mediating the primary binding between a spermatozoon and the zona pellucida (Shalgi and Raz, 1997).
Studies with 125I labeled mouse egg ZP3 and ZP2 revealed 125I-ZP3 on the acrosomal cap region of spermatozoa, and the 125I-ZP2 bound preferentially to acrosome-reacted spermatozoa (Bleil and Wassarman, 1986). Studies with anti-mouse ZP3 polyclonal antisera and monoclonal antibodies indicated that antibodies do not affect primary binding of acrosome-intact sperm to eggs or secondary binding following the acrosome reaction. This indicates that complex multiple steps that require protein of specific structure mediate the sperm to egg interaction. Unfortunately, the primary binding is difficult to detect on acrosome reacted spermatozoa, since this binding occurs on the sperm plasma membrane. A further complication is that the sperm plasma membrane fuses with the outer acrosome membrane during the acrosome reaction and is destroyed or disappears after the acrosome reaction, which signal fades away.
On the other hand, secondary binding in contrast appears stronger, more irreversible and more persistent as it is mediated by proacrosin/acrosin on the inner acrosome matrx. membrane of the acrosome-reacted spermatozoa and the ZP2 in the zona pellucida (Hyne et al., 1984; Yanagimachi, 1981). That is, a complex zona pellucida structure undergoes complex changes based on multiple specific biochemicals. However, with the ZP2 only, it cannot bind to acrosome intact spermatozoa because without acrosome reaction the inner acrosome membrane will not be exposed. The rhZP2 expressed in Escherichia coli, with two hours incubation of human spermatozoa with rhZP2 in vitro, an immunofluorescent study indicated that rhZP2 bound only to acrosome-reacted spermatozoa (Tsubamoto et al., 1999).
These detailed studies, carried out by many researchers around the world indicate that the recognition between sperm and egg is a complex and incompletely understood process. The complexity revealed by the literature in this area is an indication of the difficulty in finding a good test for diagnosis male infertility. Despite this work, a generally suitable test is lacking.
The relative lack of progress in this field is unfortunate because any mutations on the ZP2 or ZP3 genes or on the genes of membrane receptors of spermatozoa might create the binding dysfunction. Today, this binding dysfunction will not be found until these patients having been go through some infertility therapies. The infertility therapies, however, is a time and money consuming procedure. Moreover, this procedure exposes the female patients under a rugged physical and mental stress. These unnecessary sufferings should and can be avoided, if patients can access the specific therapies they need in the early stage of treatment.
The need for a high quality infertility test increases with time. During the last decade, the number of infertile couple climbed significantly and will reach 6.36 million at year 2005 in U.S. Unfortunately, although spermatozoa abnormalities are a problem in up to 40% of infertile couples (Oehninger et al., 1992), an accurate and correct test that reveals information about such sperm abnormalities is lacking. This lack impacts infertility therapy, which is a time and money consuming procedure that exposes female patients to rugged physical and mental stress. Still further, infertile couples often race with time trying to solve this problem while their inability to conceive may recede with age. Accordingly, simple and accurate sperm binding diagnostic tools are needed to guide couples into needed therapies, and will benefit infertile patients physically, emotionally and financially.